Transplant material and method for fabricating the same

ABSTRACT

A transplant material which is capable of imparting desired mechanical properties, elevating bone tissue repair speed and improving biocompatibility. This transplant material comprises an artificial and biologically inactive material, which is to be implanted in vivo as a substitute for bone tissue, and at least one type of cells selected from among osteoblasts and precursory osteoblasts which are adhered to the surface of the artificial material so that the artificial material is coated with the bone matrix produced by the cells. The artificial material involves not only a biologically inactive material but also a biologically inactive material coated with a biologically active substrate. This transplant material is produced by culturing mesenchymal stem cells collected from a living body to differentiate into at least one type of cells selected from among osteoblasts and precursory osteoblasts and then culturing the cells together with the artificial material to thereby adhere the differentiated cells on the surface of the artificial material and coat the surface of the artificial material with the bone matrix produced by the differentiated cells.

BACKGROUND OF THE INVENTION

The present invention relates to a bone tissue implant and a fabricatingmethod of the implant. The implant of the present invention can be usedas artificial joints, bones, or tooth roots, for surgical treatment ofhumans, pets, domestic animals, and the like. Specifically, it can beused as a replacement for bone tissues in a living body.

It is well known that an artificial joint is composed of a stem portion,a head portion, and an acetabular portion. The stem and head portions ofthe conventional artificial joint are composed of bioinert materials,e.g., titanium alloy, or alumina ceramics. These materials retaingreater strength and resist corrosion. The acetabular portion of theconventional artificial joint is composed of high-density polyethylene,which offers adequate resiliency and biotolerance. When the aboveartificial joint is implanted as a replacement of a damaged hip joint,the stem portion is implanted in a hollow part perforated for the upperpart of a femur through bone cement made of polymethyl methacrylate(PMMA), and the acetabular portion is implanted in a coxal bone locatedin the lower part of a pelvis through the bone cement. The implantedartificial joint sufficiently functions as a hip joint, retaininggreater strength and avoiding corrosion.

However, it is well known that the PMMA bone cement generates very highheat of polymerization when methyl methacrylate monomer is polymerizedto harden. Therefore, the problem exists that the heat of polymerizationseriously damages bone tissues where the bone tissues and bone cementcontact. To solve the above problems, a following therapy has beenperformed, which uses natural healing power of a patient. In thetherapy, the operation in which the stem portion and the acetabularportion of the artificial joint were implanted was performed so thatthese portions directly contact the bone tissues without using bonecement. Subsequently, the patient was made to keep quiet in bed untilthe artificial joint was sufficiently fixed to the bone tissues.

When the above therapy was performed, the mesenchymal stem cells whichexist in patient bone marrow migrate to the clearance between theartificial joint and the bone tissues and then are adhered.Subsequently, the adhered mesenchymal stem cells proliferate anddifferentiate to osteoblasts having high bone repair activity. Then, theosteoblasts produce bone matrix. The bone matrix covers the surfaces ofthe artificial joint and bone tissues to fill the clearance. As aresult, the artificial joint is fixed.

Japanese Laid-Open Patent Publication No. 3-45267 discloses fillingmaterials for a bone defective part and a bone vacant part. The fillingmaterials are composed of body fluid containing osteoblasts and/orosteoprogenitor cells, which is obtained from an animal, and calciumphosphate compounds. The filling materials for the bone defective partand the bone vacant part are prepared by the steps of adsorbing the bodyfluid from the animal itself to be treated to porous or granular calciumphosphate compounds, and culturing artificially, if necessary. The bonedefective part and the bone vacant part of the animal are filled withthe resultant filling materials for the bone defective part and the bonevacant part. The filling materials have excellent biocompatibility, anddo not cause xenobiotic reaction and inflammatory reaction. In addition,the leakage of the filling materials from a filling site is very little.Therefore, rapid formation of new bone may be expected.

On the other hand, the conventional artificial joint has possibility fordamaging the living tissues, such as circumferential bone tissues, sincePMMA bone cement for connecting the artificial joint to the bone tissuesgenerates a heat during the polymerization, and also the residualmonomer elutes out. Therefore, when the artificial joint is used under amechanically (i.e., physically) severe condition, the boundary facebetween the stem or acetabular portion and bone tissues surrounding itis destroyed. In addition, there is remarkably high possibility that theartificial joint will become loose or disengaged.

Further, the above-mentioned conventional therapy using natural healingpower requires a long period of time until the mesenchymal stem cells tothe surfaces of the artificial joint and bone tissues, and then producebone matrix. In particular, an aged living body requires a very longperiod of time for being completely recovered, since they have lessmesenchymal stem cells in their body and bone tissue repair speed byosteoblasts and osteoprogenitor cells is remarkably slow.

In the conventional filling materials for the bone defective part andthe bone vacant part, porous or granular calcium phosphate ceramics areused as a substrate. The ceramics composed of these substrates have lowmechanical strength, and are typically brittle. Therefore, they couldnot be used for the site in which a large load is applied or elasticdeformation is required.

BRIEF SUMMARY OF THE INVENTION

The objectives of the present invention are to solve the above problemsof the prior art; to provide implant materials, which give desiredmechanical properties as well as have improved bone tissue repair speedand biocompatibility; and to provide a method for fabricating suchimplant materials.

To achieve the above objectives, the first aspect of the presentinvention includes implant materials, which are obtained by the steps ofadhering cells to a bioinert artificial material to be implanted in aliving body as a replacement of bone tissue, wherein the cells are atleast one kind of cells selected from osteoblasts and osteoprogenitorcells; and coating the artificial material with bone matrix that isproduced by the adhered cells.

Another aspect of the present invention includes implant materials inwhich the artificial material is composed of at least one of materialsselected from titanium, titanium alloy, stainless steel, cobalt-chromiumalloy, cobalt-chromium-molybdenum alloy, alumina ceramics, carbonceramics, zirconia ceramics, silicon carbide ceramics, silicon nitrideceramics, glass ceramics, polyethylene, polystyrene,polytetrafluoroethylene, polyurethane, polyvinyl alcohol, polypropylene,polycarbonate, polymethyl methacrylate, methacrylate polymer, siliconeresin, and bioabsorbable polymer.

The further aspect of the present invention includes implant materialsin which the surface of the artificial material is coated with bioactivesubstrate.

The further aspect of the present invention includes implant materialsin which the bioactive substrate includes at least one of materialsselected from hydroxyapatite, tricalcium phosphate, calcium-deficientapatite, amorphous calcium phosphate, tetracalcium phosphate,octacalcium phosphate, fluorapatite, carbonate-apatite, calciumpyrophosphate, brushite, monetite, calcium carbonate, glass ceramicscontaining apatite, glass ceramics containing calcium metaphosphate, andbioglass.

The artificial material may be composed of at least one of metallicmaterials selected from titanium, titanium alloy, stainless steel,cobalt-chromium alloy, and cobalt-chromium-molybdenum alloy.

The artificial material may be composed of at least one of ceramicmaterials selected from alumina ceramics, carbon ceramics, zirconiaceramics, silicon carbide ceramics, silicon nitride ceramics, and glassceramics.

The artificial material may be composed of at least one of syntheticresin materials selected from polyethylene, polystyrene,polytetrafluoroethylene, polyurethane, polyvinyl alcohol, polypropylene,polycarbonate, polymethyl methacrylate, methacrylate polymer, siliconeresin, and bioabsorbable polymer.

The bone matrix may include a growth factor secreted from at least onekind of cells selected from marrow cells, mesenchymal stem cells,osteoblasts, and osteoprogenitor cells. The artificial material may beformed to have porous surface. In addition, at least part of the bonematrix may be calcified. The osteoblasts and the osteoprogenitor cellsmay be differentiated cells obtained by culturing the mesenchymal stemcells derived from a living body.

The further aspect of the present invention includes a method forfabricating implant materials of the present invention. The methodincludes steps of culturing mesenchymal stem cells obtained from aliving body to differentiate to at least one kind of cells selected fromosteoblasts, and osteoprogenitor cells, and culturing the differentiatedcells with a bioinert artificial material. As a result, thedifferentiated cells are adhered on the surface of the artificialmaterial, and the surface of the artificial material is coated with bonematrix produced by the differentiated cells.

The method may further include a step of subculturing differentiatedcells to increase the number of the cells.

Another method of the present invention includes steps of culturingmesenchymal stem cells obtained from a living body with a bioinertartificial material, adhering the mesenchymal stem cells to the surfaceof the artificial material, and differentiating the adhered mesenchymalstem cells to at least one kind of cells selected from osteoblasts andosteoprogenitor cells. As a result, the surface of the artificialmaterial is coated with bone matrix produced by the differentiatedcells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic view of an artificial joint, which is oneof the artificial materials used as an implant material of the presentinvention, having a stem portion and an artificial head portion.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present embodiments will be described below in detail.

Bone tissue is composed of bone matrix, and bone cells to whichosteoblasts and osteoprogenitor cells differentiate. The bone cells arefixed on ossepus lacunae scattered in the bone matrix. The bone matrixis composed of relatively small amount of mucopolysaccharide proteins(proteoglycan), the main component of which is chondroitin sulfate,large amount of calcium phosphate, magnesium phosphate, calciumcarbonate, and the like, and also contains various growth factors suchas bone morphogenetic protein (BMP). In addition, the bone matrixnormally contains considerable amount of collagen fibers, which givecertain degree of resiliency to the bone. On the other hand, inorganiccomponents such as apatite are formed by the action of osteoblasts(calcification of the bone matrix), which give hardness to the bone.

When the bone tissue is formed or remodeled, osteoblasts,osteoprogenitor cells, and osteoclasts, which exist at the circumferenceof the bone matrix, work, respectively. The osteoblasts and theosteoprogenitor cells are produced by the differentiation of themesenchymal stem cells. The mesenchymal stem cells exist in bone marrow,and have very vigorous differentiating potency. Dexamethasone, which isone of steroid hormones, involves in vitro differentiation of themesenchymal stem cells to osteoblasts, and osteoprogenitor cells.

The implant material of the present invention includes a bioinertartificial material implanted in a living body as a replacement of thebone tissue, which is obtained by the following steps. That is, at leastone kind of cells selected from osteoblasts and osteoprogenitor cellsare adhered to the surface of the artificial material, and theartificial material is coated with the bone matrix produced by thecells. The implant material may be used for a replacement of bone tissuewhen the bone tissue of a human, a pet, or a domestic animal aredamaged, and may be implanted in a living body.

As used herein, “implanting” is defined as when an artificiallyfabricated material is surgically implanted in a living body.

The artificial material is used in various shapes as a replacement ofthe bone tissue. For example, the artificial material may include anartificial joint such as a hip joint, a knee joint, a finger joint, ashoulder joint, an elbow joint, and an ankle joint; a metallicartificial bone; an artificial bone made of synthetic resin; anartificial bone made of ceramics; volts (screw) for coupling bonetissues; prosthesis materials; dental implant materials; and boneconnecting supplies.

The artificial materials may be composed of bioinert materials that arenot degraded or decomposed in a living body, or the decompositionproducts of which do not adversely affect on a living body. The bioinertmaterial is composed of materials other than bioactive materials, whichcan directly bond to bone tissue without using coating film (i.e.,foreign film) when implanted in a living body as a replacement of thebone tissue, and also includes biotolerant materials and bioinertmaterials.

The biotolerant material includes materials that are separated from thetissues of a living body, with connective tissue thick film (i.e.,foreign film) being formed between the bone tissues and the biotolerantmaterial when it is implanted in the living body. For example,high-density polyethylene, stainless steel, and the like may beincluded. The bioinert material includes materials in which thin film(i.e., foreign film) is intervened between the bone tissues and thebioinert material when it is implanted in a living body, or may directlyconnect a part of the bone tissues under the favorable condition. Forexample, alumina ceramics, carbon ceramics, zirconia ceramics, titaniumalloy, and the like may be included.

In addition, the artificial material is preferably composed of materialsto which mesenchymal stem cells, osteoblasts, and osteoprogenitor cellsare adhered to proliferate, and the surface of which is coated with bonematrix produced by these cells. At least one of the materials selectedfrom titanium, titanium alloy, stainless steel, cobalt-chromium alloy,cobalt-chromium-molybdenum alloy, alumina ceramics, carbon ceramics,zirconia ceramics, silicon carbide ceramics, silicon nitride ceramics,glass ceramics, polyethylene, polystyrene, polytetrafluoroethylene,polyurethane, polyvinyl alcohol, polypropylene, polycarbonate,polymethyl methacrylate, methacrylate polymer, silicone resin andbioabsorbable polymer may be included as the above-mentioned material.

When the material is used as a replacement for bone tissue that requiresgreater strength, metallic materials such as titanium, titanium alloy,stainless steel, cobalt-chromium alloy, and cobalt-chromium-molybdenumalloy, or ceramic materials such as alumina ceramics, carbon ceramics,zirconia ceramics, silicon carbide ceramics, silicon nitride ceramics,and glass ceramics may be preferable.

When the material is used as a replacement for bone tissue that requiresflexibility, elastically deformable synthetic resin materials such aspolyethylene, polystyrene, polytetrafluoroethylene, polyurethane,polyvinyl alcohol, polypropylene, polycarbonate, polymethylmethacrylate, methacrylate polymer, silicone resin, and bioabsorbablepolymer may be preferable.

The surface of the artificial material may be preferably porous. Becauseit is easy to enter mesenchymal stem cells, osteoblasts, andosteoprogenitor cells into pores of the material, and these cells aremore stably fixed in the pores.

The surface of the bioinert artificial material may preferably be coatedwith bioactive substrate. As a result, the fixation of the osteoblastsand the osteoprogenitor cells and the production of bone matrix mayfurther be promoted, and biocompatibility of the material may also beimproved. The bioactive substrate includes calcium phosphates such ashydroxyapatite, tricalcium phosphate, calcium-deficient apatite,amorphous calcium phosphate, tetracalcium phosphate, octacalciumphosphate, fluorapatite, carbonate-apatite, calcium pyrophosphate,monetite, brushite; calcium carbonates; compounds that allow to adsorbcalcium phosphates in a living body; compounds that allow to form anapatite layer on the surface of the material in a living body such asbioactive glass including glass-ceramics containing apatite,glass-ceramics containing calcium metaphosphate, and bioglass; andtitanium, titanium alloy, and polymer materials, which allow to form anapatite layer by pseudo-body fluid dipping process.

The osteoblasts or the osteoprogenitor cells may preferably be obtainedby culturing mesenchymal stem cells in a cultural solution containingdifferentiating inducing factor (dexamethasone), and differentiatingthem. Here, the mesenchymal stem cells may be proliferated by separatingand culturing marrow cells obtained from a living body, which isscheduled to be implanted the implant material. This surely preventsadverse problems such as occurrence of rejection caused by autoimmunityafter the implantation.

The surface of the artificial material may preferably be thickly coatedwith bone matrix to improve the biocompatibility after the implantation.More preferably, a part of the coated bone matrix may be calcified. Inaddition, the bone matrix may preferably contain a growth factor such asbone morphogenetic protein, which is secreted by at least one of cellsselected from marrow cells, mesenchymal stem cells, osteoblasts, andosteoprogenitor cells. Since the growth factor promotes physiologicaladhesion, proliferation, and differentiation of mesenchymal stem cellsof a living body in which the implant material was implanted, bonetissue repair speed and biocompatibility may be more improved.

The action of the implant materials will now be explained.

When the above implant material is fabricated, first an artificialmaterial having a predetermined shape, which is used for a replacementof bone tissue, is prepared. Next, the surface of the artificialmaterial is coated with bioactive substrate, such as hydroxyapatiteusing plasma spraying process, pseudo-body fluid dipping process,alternate dipping process, and the like. Subsequently, the coatedartificial material is sterilized. Next, marrow cells are obtained usinga syringe from a living body that is scheduled to be implanted. At thisstage, it is preferable to increase the number of mesenchymal stem cellsobtained from the marrow cells through well-known separating, culturing,or subculturing techniques.

Subsequently, the marrow cells or the mesenchymal stem cells are adheredto the artificial material, and the resultant material is cultured in asolution containing dexamethasone, β-sodium glycerophosphate, andascorbic acid, and the cells adhered to the artificial material aredifferentiated to osteoblasts and osteoprogenitor cells. Then, thesurface of the artificial material is coated with bone matrix producedby these cells. Alternatively, the marrow cells or the mesenchymal stemcells may be cultured in the culture solution in advance todifferentiate to osteoblasts and osteoprogenitor cells. The resultantosteoblasts and osteoprogenitor cells may be cultured with a sterilizedartificial material. As a result, these cells may adhere to the surfaceof the artificial material, and the surface of the artificial materialmay be coated with bone matrix produced by these cells.

When the marrow cells or the mesenchymal stem cells are cultured withthe artificial material to differentiate, a cell-suspended solutioncontaining the marrow cells or the mesenchymal stem cells is prepared,and then an artificial material is soaked in the cell-suspendedsolution. As a result, the cells are adhered to the surface of theartificial material soaked in the solution. Subsequently, the artificialmaterial is cultured in the solution. The marrow cells or themesenchymal stem cells adhered to the artificial material proliferate atthe surface of the artificial material, and then differentiate toosteoblasts and osteoprogenitor cells. The differentiated osteoblastsand osteoprogenitor cells extracellularly produce bone matrix. Thesurface of the artificial material is directly coated with the bonematrix without being intervened with a foreign film.

Alternatively, when the marrow cells or the mesenchymal stem cells aredifferentiated to the osteoblasts and the osteoprogenitor cells inadvance, and then the differentiated cells are cultured with theartificial material, the marrow cells or the mesenchymal stem cells areinitially cultured in a culture dish, which fills with the culturesolution. These cells quickly proliferate while adhering on the bottomsurface of the culture dish since they have high proliferation potency.Subsequently, when these cells are cultured in the culture solution,they are differentiated to a large number of osteoprogenitor cells andosteoblasts. If necessary, subculture is performed to additionallyincrease the number of cells. After the number of the osteoblasts andthe osteoprogenitor cells sufficiently increases, these cells aredetached from the bottom surface of the dish using trypsin solution orthe like, and then suspended in the culture solution to prepare acell-suspended solution. Subsequently, the artificial material is soakedin the cell-suspended solution, and then incubated in an incubator.Here, some osteoblasts and osteoprogenitor cells adhere to the surfaceof the artificial material soaked in culture solution, and then grow onits surface. Then, the cells produce bone matrix. The resultant bonematrix is directly coated on the surface of the artificial materialwithout a foreign film being intervened.

When the above implant material is implanted in a living body as areplacement of bone tissue, the surface of the implant material iscoated with cells of a living body itself and bone matrix derived fromthe living body to be implanted. Therefore, the implanted material hasconsiderably high biocompatibility for adjacent bone tissues, othertissues, and cells. In addition, since the bone matrix is coated on anentire surface of the implant material, the living body does notrecognize implant material as a foreign body, and occurrence ofinflammatory reaction and formation of coated film (i.e., foreign film)can be avoided.

In addition, the osteoblasts and the osteoprogenitor cells that areadhered to the surface of the implant material in advance have alreadyhigh bone repair activity at the time when implanted. Therefore, newbone formation is started while the circumference of the implantedmaterial is coated with the bone matrix immediately after theimplantation. With the passage of the time, the clearance between theimplant material and bone tissues surrounding the implant material isminutely filled with the cells to repair the bone, and then the implantmaterial is more surely fixed.

Also, the bone matrix produced on the surface of the implant materialpromotes physiological adhesion and growth of the pluripotentmesenchymal stem cells, osteoblasts, and osteoprogenitor cells of theliving body. Therefore, these cells are physiologically adhered to theimplant material after implantation. These cells start the bone repairprocess and new bone matrix is deposited to the implant material. Thisachieve reliable fixation of the implant material in the bone tissue.

After additional period of time passes, repaired bone tissues thatsurround the implant material would continually maintain the conditionthat is always and properly renewed by the physiological metabolism ofthe living body through the work of the osteoclasts, the osteoprogenitorcells, and the osteoblasts. If this condition is maintained, eitherlooseness or disengagement of the implant material rarely occurs for along time use.

The above embodiments of the present invention have the followingeffects.

The implant material of the present embodiment is obtained by the stepsof adhering at least one kind of cells selected from osteoblasts andosteoprogenitor cells to the surface of a bioinert artificial material,which is implanted in a living body as a replacement of bone tissue, andcoating with bone matrix produced by the adhered cells. Since thebioinert material is used as the artificial material, it is possible toeasily and surely give desired mechanical properties (mechanicalstrength, abrasion resistance, and the like) for an implant material.That is, the implant material may be selected based on wide variety ofusages, such as a replacement for bone tissues having high mechanicalstrength, bone tissues having elastic deformability, and the like.

Since the cells having greater bone repair ability are adhered to thesurface of the implant material, they fill the clearance between theimplant material and bone tissues surrounding the implant material fromimmediately after the implantation, and the speed of bone tissue repairis increased. There are many cases in which bone tissue repair speed invivo is slow in an aged organism. Even in such a case, early treatmentand fixation of the implant material may surely be achieved by adheringcells with artificially enhanced bone repair activity. Therefore, thetime required for complete recovery may be reduced, the period forincurring the bodily and mental suffering may also be reduced, and thecost for the treatment may be reduced.

In addition, since the bone matrix produced by the osteoblasts and theosteoprogenitor cells are coated on the surface of the implant material,the biocompatibility may be improved without a film (a foreign film)being formed between surface of the implant material and bone tissuessurrounding the implant material. Therefore, it is possible to maintainhigh biocompatibility of the implant material in vivo for a long periodof time from immediately after the implantation. Also, physiologicaladhesion and growth of pluripotent mesenchymal stem cells, osteoblasts,and osteoprogenitor cells in vivo are facilitated.

As mentioned above, according to the present invention, early treatmentand early fixation of the implant material to the bone tissues areeasily and surely achieved. In addition, the implanted material may beused for a long period of time without any troubles.

In the conventional artificial joint, PMMA bone cement or an artificialmaterial, which is a bioinert material, directly contacts the bonetissues. Therefore, there was a remarkably high possibility that filmwas formed on the surface of the artificial joint and the failure, suchas looseness or disengagement of the artificial joint, occurs. However,the implant material of the present embodiments drastically raises thebiocompatibility due to the bone matrix coated on the surface of theimplant material. As the result, looseness and disengagement of theartificial joint rarely happen, and the implanted material may use for along period of time without any troubles.

In addition, since the PMMA bone cement generates very high heat ofpolymerization, the bone tissue receives serious damages when contactingthe bone cement. As the result, the biocompatibility between bone cementand bone tissues surrounding the artificial joint drastically lowered,and the burden for a living body remarkably increased. In contrast, theimplant material of the present embodiments has very much increased bonerepair activity. Therefore, without using bone cement, the earlyfixation of the implant material can be achieved using the naturalhealing power of a living body. Therefore, the above problems of heat ofpolymerization may be easily and surely solved. In addition, theproblems of harmful residual monomer, which occur during thepolymerization of the PMMA bone cement, may be surely solved.

In the conventional implant material such as an artificial joint, it hasbeen studied that the bone repair activity was raised by administeringbiological factors such as cytokine, which is an expensive drug in thetreatment. On the other hand, since osteoblasts and osteoprogenitorcells having raised bone repair activity are adhered to the implantmaterial of the present embodiments, and since it is not necessary toadministrate the above biological factor, the present embodiments areeconomical. In addition, the burden for post-operative care may bealleviated.

Conventionally, bone autograft substitute has been performed, in whichbone tissue such as an iliac bone is obtained from a living body that isscheduled to be implanted and then is implanted into the living body asa replacement for damaged bone tissue. However, an extra operation isnecessary for obtaining bone tissue other than damaged tissue in thisbone autograft substitute. On the other hand, according to the presentinvention, it is only necessary to perform an operation with smallburden in which marrow cells are obtained using a syringe. Therefore,the above extra operation is not necessary, and the physical and mentalsuffering of the living body to be implanted may also be reduced.

Bone matrix of the implant material may further include biologicalfactor, such as growth factor secreted from at least one kind of cellsselected from marrow cells, mesenchymal stem cells, osteoblasts, andosteoprogenitor cells. As a result, physiological adhesion andproliferation of mesenchymal stem cells of a living body in which theimplant material was implanted and differentiation to osteoblasts andosteoprogenitor cells are promoted. In addition, even if cells adheredto the surface of the implant material are accidentally dead, bonematrix and the biological factor contained in the bone matrix promotesthe physiological adhesion of the mesenchymal stem cells existing in theimplanted living body. Therefore, bone tissue repair speed andbiocompatibility are enhanced.

The artificial material is composed of at least one of materialsselected from titanium, titanium alloy, stainless steel, cobalt-chromiumalloy, cobalt-chromium-molybdenum alloy, alumina ceramics, carbonceramics, zirconia ceramics, silicon carbide ceramics, silicon nitrideceramics, glass ceramics, polyethylene, polystyrene,polytetrafluoroethylene, polyurethane, polyvinyl alcohol, polypropylene,polycarbonate, polymethyl methacrylate, methacrylate polymer, siliconeresin and bioabsorbable polymer. As a result, degradation ordecomposition of the implant material in vivo, or the adverse effects ofthe decomposed products from the implant material on a living body areavoided, and sure role as a replacement for damaged bone tissue isperformed.

In addition, since most suitable material for treatment is properlyselected from a wide variety of the materials, an implant materialhaving desired mechanical property is obtained. More particularly, sincethe artificial material composed of metallic material and ceramicmaterial has high mechanical strength, it is suitable for a replacementof bone tissue that requires high mechanical strength. Artificialmaterial made of ceramics is made to easily form one having poroussurface. Therefore, a weight saving of an implant material may beachieved. Artificial material made of elastically deformable syntheticresin is suitable for a replacement of the bone tissue that requiresflexibility.

Bioinert artificial material may promote implantation, growth, and bonerepair of mesenchymal stem cells, osteoblasts, and osteoprogenitorcells, when the bioactive substrate is coated on the surface of theartificial material.

It is possible to surely prevent the rejection by the autoimmunity of animplanted living body by using, as cells to be adhered to the implantmaterial, osteoblasts and osteoprogenitor cells obtained bydifferentiating cultured mesenchymal stem cells derived from a livingbody scheduled to be implanted.

Even if the mesenchymal stem cells are those obtained from an agedliving body, it is possible to culture the cells in vitro to proliferatethem. As a result, they have almost the same proliferating anddifferentiating potency, as well as bone repair activity, as thosederived from a young living body, and excellent therapeutic effects areachieved in the aged living body.

By designing the surface of an artificial material to be porous, it ispossible that mesenchymal stem cells, osteoblasts, and osteoprogenitorcells easily enter the pores of the artificial material, and these cellsare fixed in a stable condition. Since the superficial area increases inthe porous artificial material, it is possible to fix more cells to theimplant material. It is also possible to avoid looseness anddisengagement of the implant material, since the larger area and themore complicated shape of the porous implant material contact bonetissues surrounding the implant material after the bone repairing.

Calcification of the bone matrix achieves very firm bonding between theimplant material and the bone tissues surrounding the implant material.Accordingly, biocompatibility of the implant material is more enhanced,and the period until complete recovery is remarkably reduced.

The method for fabricating the implant material according to the presentembodiments includes the steps of culturing mesenchymal stem cellsobtained from a living body to differentiate to at least one kind ofcells selected from osteoblasts and osteoprogenitor cells, and culturingthe differentiated cells with a bioinert artificial material. As aresult, the differentiated cells are adhered to the surface of theartificial material, and the surface of the artificial material iscoated with bone matrix produced by the differentiated cells. The abovemethod easily achieves the production of the implant material having thedesired mechanical property and improved bone tissue repair speed andbiocompatibility.

EXAMPLES

The present invention will now be described with reference to thefollowing examples in which the above-mentioned embodiments areembodied, and comparative examples.

Subcutaneous Implantation Experiment Example 1

Marrow cells were obtained from a bone shaft of femurs of 7 week-oldmale Fischer rats, and α-MEM (minimum essential medium) containing 15%of fetal bovine serum (FBS) was added to the cells, and then obtainedculture was primarily cultivated in an incubator (37° C., an atmosphereof 5% CO₂) for 7–12 days.

After the primary culture, the cultured marrow cells were treated with0.01% of trypsin solution, and 1×10⁶ to 1×10⁷ cells/ml of cellsuspension was prepared. Disk artificial materials having a diameter of34 mm and a thickness of 2 mm (made of titanium alloy, stainless steel,alumina ceramics and high-density polyethylene) were soaked in thesuspension, and were incubated in an incubator for two hours (37° C., anatmosphere of 5% CO₂). Porous artificial materials having a rectangularparallelepiped shape (3×3×5 mm) (made of titanium alloy, stainlesssteel, alumina ceramics and high-density polyethylene) were soaked inthe suspension, and were incubated in an incubator for about 2 hours, asdescribed in the above procedure.

Subsequently, these materials were transferred to a 35 mm culture dish,to which 2 ml of medium containing antibiotics, 10⁻⁸ M of dexamethasone,10 mM of β-sodium glycerophosphate, and 50 μg/ml of ascorbic acid wasadded, and were incubated for about one week (37° C., an atmosphere of5% CO₂). If necessary, the medium was changed.

It was confirmed by alkaline phosphatase activity and alizarin red stainthat the osteoprogenitor cells and the osteoblasts differentiated fromthe mesenchymal stem cells derived from bone marrow were adhered to thesurface of the implant material, and the surface thereof was coated withbone matrix produced by these cells. In addition, the experiment inwhich the porous implant material was implanted in the back hypodermicof syngeneic rats was performed. About one to two weeks later, new boneformation was confirmed on the surface of the implant material.

Comparative Example 1

Four kinds of artificial materials (titanium alloy, stainless steel,alumina ceramics, and high-density polyethylene) were directly implantedin the back hypodermic of the rats. As a result, the intervention offibrous tissue (a foreign film) surrounding the artificial materials wasconfirmed in the early stage after the implantation, and new boneformation was never confirmed.

Artificial Joint Using Test Example 2

Marrow cells were obtained from an adult dog, and were primarilycultured for 7–12 days, as is like Example 1. After cultivation, acell-suspended solution was prepared. The stem portion of the artificialhip joint composed of titanium alloy was soaked in the cell-suspendedsolution, and was incubated for two hours (37° C., an atmosphere of 5%CO₂) Subsequently, the stem portion was transferred to a culturecontainer containing culture medium identical to the medium used inexample 1, and was cultured in an incubator (37° C., an atmosphere of 5%CO₂) for about 1 week. If necessary, the medium was changed.

It was confirmed that the osteoprogenitor cells and the osteoblastsdifferentiated from the mesenchymal stem cells derived from bone marrowwere adhered to the surface of the implant material, and the surfacethereof was coated with bone matrix produced by these cells. Inaddition, when the stem portion of the artificial hip joint wasimplanted into a cavity of the femur of the adult dog, fixation betweenthe stem portion and the femur was confirmed. In the artificial hipjoint, new bone formation was confirmed on the surface of the stemportion in early stage after the implantation, and the fixation of thestem was also quickly finished.

Comparative Example 2

An artificial hip joint without being soaked in the cell-suspendedsolution was implanted as is like in example 2. As a result, partialfixation between the stem portion and the femur was observed, and wasresulted in the looseness of the stem portion.

Example 3

2 ml of marrow cells were obtained from a humerus of a beagle dog having12 kg of body weight. The obtained marrow cells were transferred to atube containing 2 ml of FBS with heparin, and were centrifuged (900 rpm,10 minutes, 24° C.) After fat cells and supernatant were removed fromthe centrifuged tube, cells were transferred to a T-75 flask, and wereprimary-cultured in a medium containing 15% of FBS and antibiotics forten days. The culture medium was exchanged 3 times per week.

In this embodiment, an implant material having a stem 1 made of titanium(hereafter referred as “Ti”) and an artificial head 2 made of highpurity alumina were used as shown in FIG. 1. Both surfaces of the stem 1include associated recesses 3 (depth; 0.5 mm, area; 1.3 cm²). Theimplant material was treated with thermal spraying processing of pureTi. Average surface roughness of the thermal spraying plane was 32 μm.After the primary culture, 0.25% of trypsin was used for detachmentprocessing, and a cell-suspended solution was prepared.

A stem for a dog was put in the culture dish having a 94 mm diameter,and 1×10⁵ cells of the cell-suspended solution was put on a surface tobe treated with the pure Ti thermal spraying processing (area; 1.3 cm²)of upper stem side, and incubated at 37° C. for 30 minutes to adhere thecells to the surface to be treated with the Ti thermal sprayingprocessing. Subsequently, 48 ml of medium containing 10 mM ofβ-glycerophosphate, 82 μg/ml of vitamin C phosphate, and 10⁻⁸ M ofdexamethasone was added to carry out the subculture for 11 days.

When subcultured stem was stained using a stain for an alkalinephosphatase, a surface treated with Ti thermal spraying processing inwhich the cells were seeded was stained in red. On the other hand, asurface treated with Ti thermal spraying processing in which the cellswere not seeded, i.e., the rear side of the stem, was not stained. Thismeans that cells differentiated to the osteoblasts existed on an entiresurface in which the cells were seeded.

A line from greater trochanter to lesser trochanter at femur head sideof a right femur of a beagle dog was made to incision, and the femurhead was extracted. Rasping was performed using medullary cavityexpansion and trial. Surfaces of the stem, in which the above preparedcells were mounted on one side, was washed with PBS (−) andphysiological saline solution. Subsequently, the stem was inserted intoa medullary cavity of the femur, and an artificial bone head wasattached to the stem.

The dog was sacrificed three weeks after the operation, and theimplanted stem portion was extracted. The surface of the stem, which istreated with the Ti thermal spraying processing, was histologicallyobserved. The Ti spraying surface in which the cells were mounted wasobserved that new bone was formed in the cavity of the Ti sprayingsurface, the entire surface in which the cells were mounted was coveredwith the new bone. On the contrary, the Ti spraying surface in which thecells were not mounted was observed that the stem was partiallycontacted with the bone.

The present embodiments may be embodied with following modifications.

The artificial material may be composed of the combination of metallicmaterial or ceramic material with synthetic resin. For example, in theartificial joint consisting of a stem portion, a head portion, and anacetabular portion, the stem portion and the head portion may be made ofthe metallic material or the ceramic material, while the acetabularportion may be made of the synthetic resin material. The abovecomposition achieves an implant material in which each part has itsoptimal mechanical properties. Therefore, it is possible to reducediscomfort for a living body.

The surface of an implant material made of bioinert material may not becoated with bioactive substrate. Even when the above implant material isused, the mesenchymal stem cells, osteoblasts, and osteoprogenitor cellsmay be adhered to the surface of the artificial material, and thesurface of the artificial material may be coated with bone matrixproduced by the osteoblasts and the osteoprogenitor cells.

The surface of the artificial material may not be formed porous, but beflat. The artificial material with a flat surface may easily be molded.

The implant material may be constituted of an artificial material thatincludes bioinert material in which mesenchymal stem cells, osteoblasts,and osteoprogenitor cells could not be adhered and proliferated, and iscoated with a bioactive substrate on the surface of the artificialmaterial. The above implant material may allow to adhere at least onekind of cells selected from the osteoblasts and the osteoprogenitorcells to the surface of the implant material, and may be coated with thebone matrix.

As an osteoblast or a osteoprogenitor cell to be adhered to the surfaceof the implant material, cells derived from homogeneous biologicalorganisms having identical major histocompatibility complex (MHC), orhuman lymphocyte antigen (HLA) may be used. Alternatively, cells that donot have identical MHC or HLA may be used, while the immunosuppressantmay be administered. When the above cells are used for an implantmaterial, it is possible to surely suppress the rejection byautoimmunity. When marrow cells of a living body scheduled to beimplanted are suspicious of cancerous, it is afraid that innidiation andmetastasis of the cancer is promoted by implanting an implant materialin which cells derived from the cancerous marrow cells are adhered tosuch a living body. However, the above possibility may be avoided byusing marrow cells obtained from a healthy, non-cancerous organism. Inaddition, when a cord blood, more specifically, the mesenchymal stemcells included in the cord blood may be used, it is very advantageous.

A part of the mallow cells or the mesenchymal stem cells may be culturedin the cultural solution with the artificial material, and the remaindermay be cultured on the culture dish. After the number of the cells onthe culture dish increases, the cells may be adhered to the surface ofthe artificial material, and then cultured. In addition, a part ofcells, the number of which is increased by the subculture, may beadhered to the surface of the artificial material and be cultured, andthe remaining cells may further be subcultured. After the number of thesubcultured cells increases, the subcultured cells may be adhered to thesurface of the artificial material, and be cultured. The aboveconstruction may efficiently increase the number of osteoblasts andosteoprogenitor cells in a short period of time. This greatly shortensthe period for fabricating the implant material. In addition, more cellsmay be adhered to the surface of the implant material.

The implant material may be fabricated using an artificial materialmolded on the shape that conforms to a bone defective part or a bonevacant part, and the resultant implant material may be implanted on thebone defective part or the bone vacant part. The above implant materialmay give the desired mechanical properties, and improve the bone tissuerepair speed and the biocompatibility.

As a differentiating inducing factor for differentiating mesenchymalstem cells to osteoblasts and osteoprogenitor cells, bone morphogeneticprotein, fibroblast growth factor, glucocorticoid or prostaglandin maybe used.

At least one growth factor selected from dexamethasone, bonemorphogenetic protein, fibroblast growth factor, glucocorticoid, andprostaglandin may be attached to, adhered to, or soaked in the surfaceof the implant material. The above implant material may further beenhanced its bone repair activity.

1. A bone tissue implant comprising a bioinert artificial material andosteoblast cells or non-embryonic osteoprogenitor cells, adhereddirectly on the material, whereby a bone matrix produced by the cells invitro coats the material.
 2. The implant according to claim 1, whereinthe material comprises titanium, titanium alloy, stainless steel,cobalt-chromium alloy, cobalt-chromium-molybdenum alloy, aluminaceramics, carbon ceramics, zirconia ceramics, silicon carbide ceramics,silicon nitride ceramics, glass ceramics, polyethylene, polystyrene,polytetrafluoroethylene, polyurethane, polyvinyl alcohol, polypropylene,polycarbonate, polymethyl methacrylate, methacrylate polymer, siliconeresin, or a bioabsorbable polymer.
 3. The implant according to claim 1,wherein the material comprises titanium, titanium alloy, stainlesssteel, cobalt-chromium alloy, or cobalt-chromium-molybdenum alloy. 4.The implant according to claim 1, wherein the material comprises aluminaceramics, carbon ceramics, zirconia ceramics, silicon carbide ceramics,silicon nitride ceramics, or glass ceramics.
 5. The implant according toclaim 1, wherein the material comprises polyethylene, polystyrene,polytetrafluoroethylene, polyurethane, polyvinyl alcohol, polypropylene,polycarbonate, polymethyl methacrylate, methacrylate polymer, siliconeresin, or a bioabsorbable polymer.
 6. The implant according to claim 1,wherein the bone matrix comprises a growth factor secreted from marrowcells, mesenchymal stem cells, osteoblast cells, or osteoprogenitorcells.
 7. The implant according to claim 1, wherein the materialcomprises a porous surface.
 8. The implant according to claim 1, whereinat least part of the bone matrix is calcified.
 9. The implant accordingto claim 1, wherein the cells are obtained by culturing and thendifferentiating the mesenchymal stem cells derived from a living body.